The invention relates to a family of fluorescent labeled conjugates of sulforhodamine 101, and the chemically reactive fluorescent dyes that are used to prepare those conjugates, including a wide range of biologically-derived or synthetic chemical materials.
Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Fluorescent dyes are used to impart both visible color and fluorescence to other materials. Dyes that are able to preferentially bind to a specific biological ingredient in a sample enable the researcher to determine the presence or quantity of that specific ingredient. In addition, specific cellular structures can be monitored with respect to their spatial and temporal distribution in diverse environments. Many applications utilize chemically reactive fluorescent dyes by chemically attaching the dye to reactive sites on a wide variety of materials such as cells, tissues, proteins, antibodies, enzymes, drugs, hormones, lipids, nucleotides, nucleic acids, or natural or synthetic polymers to make fluorescent conjugates.
Reactive derivatives of a wide variety of fluorescent dyes have been previously described. This family of reactive dyes includes the sulfonyl chloride derivative of sulforhodamine 101 (Titus et al. J. IMMUNOL. METH., 50, 193 (1982)), sold by Molecular Probes, Inc. under its registered trademark TEXAS RED. The sulfonyl chloride of sulforhodamine 101 (hereafter referred to as SSC) is typically available as a mixture of isomers, as shown below: 
Conjugates of SSC with proteins and other biomolecules have achieved wide acceptance, particularly because their red fluorescence is readily distinguishable from that of fluorescein (FIG. 2), with which they are often combined in assays (Titus et al., supra).
Despite its many favorable characteristics, SSC possesses several disadvantages when used as a protein label. It is difficult to prepare protein conjugates that have a high degree of SSC-labeling due to the strong tendency of the resulting SSC-conjugates to precipitate from solution (Titus et al., supra; Waggoner et al., PRINCIPLES OF CLINICAL FLOW CYTOMETRY, Chapter 7, pp 111-116). Furthermore, SSC is not appreciably soluble in water, necessitating prior dissolution of SSC in an organic solvent, which must then be added to the protein. In addition, the short spacer between SSC and the attachment site makes SSC-conjugates more likely to hinder the labeled probe in biological interactions, such as with an enzyme, as when the SSC-labeled nucleotides are used in conjunction with a nucleotide polymerase, or with a ligand binding site, as in the case of a drug receptor. Finally, the fluorescence emission of SSC-labeled proteins tends to be quenched, relative to the fluorescence emission of the free dye.
In addition, the SSC dye itself possesses a sulfonyl chloride reactive site, so it is intrinsically very susceptible to hydrolysis by even trace amounts of water. This instability often results in labeling variability, or total labeling failure. Sulfonyl chlorides also react non-selectively with groups in proteins other than amines, including tyrosine, histidine and serine residues. Where these residues are essential for biological activity of the proteins, this reactivity is detrimental to the use of the labeled protein. Furthermore, instability of these undesired adducts results in slow loss of the dye from the conjugates during storage.
The modification of SSC to form the dyes of the present invention allows the use of other less problematic reactive groups on the fluorophore, allowing conjugation with a wider range of substances. Derivatives of sulforhodamine 101 that selectively react with functional groups other than amines (e.g. thiol-reactive dyes) were previously unknown. An aminocaproic acid derivative of sulforhodamine 101 has recently been described in the literature (Abuelyaman et al., BIOCONJUGATE CHEM. 5, 400 (1994)), used as the hydroxybenzotriazole ester to prepare only a particular sulforhodamine 101-labeled peptide phosphonate. The resulting intermediate activated ester is less chemically stable than analogous N-hydroxysuccinimidyl esters. Succinimidyl esters of aminoalkanoic acids are known for other fluorophores, including tetramethylrhodamine (Haugland, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, (1992)) and lissamine rhodamine (U.S. Pat. No. 5,393,514 to Pitner et al., (1995)). These fluorophores possess a shorter wavelength than those of the present invention, and they do not possess all of the advantages of the reactive dyes of the invention, in particular, a fluorescence emission that is well separated from that of fluorescein.
The novel dyes of the invention are derived from SSC, but they possess many advantages that overcome the cited limitations of SSC, including greater water solubility, selective reactivity with a broader range of functional groups, and considerably enhanced stability relative to SSC. The reactive dyes have sufficient chemical stability to permit their purification to a high degree of purity, including their separation into chemically pure single isomers, and the removal of trace amounts of disulfonyl chloride impurity, which is not practical for the hydrolytically unstable SSC. In addition, the conjugates that result from the use of the new dyes retain or improve upon the beneficial properties of labeling with sulforhodamine 101. Selected embodiments of the invention react with, and form conjugates with, substances having reactive functional groups, including amines, thiols, alcohols and phenols. The conjugates of the present invention have fluorescence emission wavelengths that are the same or longer in wavelength than those prepared from SSC, and thus have even less spectral overlap with the emission of fluorescein (FIG. 2). Furthermore, the conjugates prepared using the dyes of the present invention typically possess a higher fluorescence quantum yield than SSC-conjugates, both as the result of permitting a higher degree of substitution, and an unexpected reduction of quenching of the dyes by their conjugates (FIG. 7). The protein-conjugates of the present invention are unexpectedly much more soluble than those prepared using SSC, and are less prone to precipitation. Finally, the dyes of the present invention label oligonucleotides more efficiently than SSC, and the resulting oligonucleotide conjugates are more readily purified.